Project Summary The HIV-1 genome contains highly conserved and structured regulatory RNA elements that offer unique opportunities to inhibit viral replication in a manner that is potentially less prone to resistance-conferring mutations as compared to other protein targets. In vivo, the most attractive HIV-1 RNA drug targets such as TAR and RRE bind to protein assemblies through large interaction surfaces and strong electrostatic forces. Inhibiting such RNA-protein interactions using a small molecule presents a significant challenge. In addition, the structure of TAR and other HIV-1 RNA drug targets are dominated by canonical Watson-Crick helices that form shallow solvent-exposed binding pockets and limited structural uniqueness. Similar structures can be found across the human transcriptome. Compounds that bind to these RNAs frequently bind to other related RNAs and exhibit weak selectivity and/or poor pharmacological properties. The goal of this project is to advance a new anti-HIV therapeutic strategy involving inhibition of Tat-dependent transcriptional activation through targeted stabilization of an inactive conformation of the transactivation response element (TAR) RNA. The long-term goal of this proposal is to identify drug-like small molecules that selectively bind and stabilize this inactive TAR conformation, thereby allosterically inhibiting transcriptional activation and HIV replication. Aim 1 will apply ensemble-based virtual screening to guide experimetnal screening of 1 million compounds to identify inhibitors of transcriptional activation and HIV replication that selectively bind TAR and bias the conformtional equilbrium toward the alternative excited state. Aim 2 will apply structure- based approaches to further optimize current hits as well as other hits generated in Aim 1 through organic synthesis. If successful, this work will develop a new class of inhibitors of HIV-1 transcription activation and potentially lay out a new paradigm for targeting RNA based on stabilization of excited states that can be broadly applied to treat a wide range of diseases.